宫颈癌细胞中NRDR选择性剪接新亚型表达质粒的构建及原核表达

Expression and Purification of NADP(H)-dependent Retinol Dehydrogenase/reductase (NRDR) in Cervical Carcinoma

背景与目的：构建宫颈癌细胞中视黄醇脱氢／还原酶．辅酶Ⅱ依赖性视黄醇脱氢／还原酶（NRDR）及其选择性剪接亚型NRDRB1原核表达载体，并在BL21-AI大肠杆菌中表达融合蛋白。材料与方法：用RT-PCR检测宫颈癌组织中NRDR、NRDRB1表达，RACE方法克隆NRDRB1全长eDNA，运用Gateway表达系统将NRDR、NRDRB1编码区序列构建到表达载体，转化到大肠杆菌中表达，表达的融合蛋白通过金属离子亲和层析纯化。结果：在宫颈鳞癌组织中发现NRDR新的选择性剪接亚型NRDRBl，构建了NRDR、NRDRBl原核表达载体，在BL21-AI中表达出氨基端含6×His标签的重组蛋白，诱导表达4h后目的蛋白可占总蛋白量的30％．50％，经一步亲和层析得到了高纯度的NRDR及NRDRB1重组蛋白。结论：在大肠杆菌中获得高效表达的NRDR、NRDRB1蛋白，为进一步研究其功能提供了实验材料。

BACKGROUND AND AIM： To express NRDRB1 in prokaryotic expression system for detecting enzyme activity and preparing polyclonal antibody. MATERIALS AND METHODS： We investigated the mRNA expression of NRDRB1 in cervical squamous carcinoma using RT-PCR. The total NRDR and NRDRB1 sequences were cloned and the coding regions were constructed to the Gateway-based expression vector （pDEST 17）, which was transformed into the Escherichia coli（BL21-AI） for protein expression. The recombinant proteins were purified by affinity chromatography. RESULTS： We identified a novel alternatively spliced variant, NRDRB1, in HeLa cell and human cervical squamous carcinoma cells, characterized by a complete deletion of exon 3. The expression vectors of NRDR and NRDRB1 were constructed. The proteins, harvested at the optimal time point （4 hours after induction）, were successfully expressed with a 30-50% expression level. The homogeneous proteins were obtained by a one-step affinity chromatography. CONCLUSION： Recombinant NRDR and NRDRB1 from human cervical squamous carcinoma cells were expressed effectively in BL21-AI by pDEST 17.